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Introduction: Imaging studies patients with mitral valve prolapse (MVP) have previously shown cross sectional evidence of left ventricular (LV) fibrosis as well as increased risk for sudden cardiac death. This, along with evidence for post-repair ventricular dysfunction in Mitral valve surgery patients support a link between fibrosis and MVP. The development of fibrosis as a result of MVP, however, remains poorly understood. Hypothesis: Regionalized LV Fibrosis in MVP patients is driven by increased mechanical tension through the chordae tendineae by billowing leaflets. Methods: We performed histopathologic analysis on cardiac biopsies of 6 patients who underwent surgical repair for MV prolapse. We additionally performed longitudinal histopathologic analysis on hearts harvested from a mouse modal of human non-syndromic MVP (Dzip1 S14R/+). To assess the cellular response to increased mechanical tension, pathological stretch assays on human cardiac fibroblasts were performed in tandem with RNA seq, as well ATP luminescence assays to assess ATP release from stretched cells. Results: Biopsies from peripapillary regions, inferobasal wall, LV septum, and LV apex demonstrated regionalized myocardial fibrosis that correlated with increased macrophages and myofibroblasts. Our mouse model demonstrated similar regionalized deposition of collagen that increased over time, as well as an increase in macrophages and myofibroblasts. Stretching of human cardiac fibroblasts caused significant changes in 232 transcripts (Bonferroni corrected P value Conclusions: A mouse model of human MVP phenocopied regionalized myocardial fibrosis seen in humans and will provide a useful tool to investigate mechanisms of myocardial fibrosis in MVP patients. Stretch initiates changes in pro-fibrotic gene transcription, and causes ATP release from human cardiac fibroblasts.

Background: Bicuspid aortic valve (BAV) disease is a congenital defect that affects 0.5% to 1.2% of the population and is associated with comorbidities including ascending aortic dilation and calcific aortic valve stenosis. To date, although a few causal genes have been identified, the genetic basis for the vast majority of BAV cases remains unknown, likely pointing to complex genetic heterogeneity underlying this phenotype. Identifying genetic pathways versus individual gene variants may provide an avenue for uncovering additional BAV causes and consequent comorbidities. Methods: We performed genome-wide association Discovery and Replication Studies using cohorts of 2131 patients with BAV and 2728 control patients, respectively, which identified primary cilia genes as associated with the BAV phenotype. Genome-wide association study hits were prioritized based on P value and validated through in vivo loss of function and rescue experiments, 3-dimensional immunohistochemistry, histology, and morphometric analyses during aortic valve morphogenesis and in aged animals in multiple species. Consequences of these genetic perturbations on cilia-dependent pathways were analyzed by Western and immunohistochemistry analyses, and assessment of aortic valve and cardiac function were determined by echocardiography. Results: Genome-wide association study hits revealed an association between BAV and genetic variation in human primary cilia. The most associated single-nucleotide polymorphisms were identified in or near genes that are important in regulating ciliogenesis through the exocyst, a shuttling complex that chaperones cilia cargo to the membrane. Genetic dismantling of the exocyst resulted in impaired ciliogenesis, disrupted ciliogenic signaling and a spectrum of cardiac defects in zebrafish, and aortic valve defects including BAV, valvular stenosis, and valvular calcification in murine models. Conclusions: These data support the exocyst as required for normal ciliogenesis during aortic valve morphogenesis and implicate disruption of ciliogenesis and its downstream pathways as contributory to BAV and associated comorbidities in humans.

Introduction: Mitral valve prolapse (MVP) is a major source of morbidity and mortality and is becoming one of the most common indications for cardiac surgery. Our group has previously reported that MVP is associated with missense mutations in DCHS1 , a gene widely implicated in tissue development and organization. Purpose: We recently performed two-hybrid screens and co-immunoprecipitation assays that define a novel complex between DCHS1, LIX1L, and SEPT9 (DLS). Our current studies investigate the role of this interaction in mitral valve morphogenesis. Methods: Wild-type (WT), DCHS1, and/or LIX1L heterozygous neonate murine heart tissue was isolated for primary culture of cardiac fibroblasts (CFs) and analyzed with immunocytochemistry, collagen, compaction, fibrin pillar to post assays, and actin sedimentation assays. Nuclear morphology and chromatin density were conducted for functional read-outs of cytoskeletal aberrations. Morphologic and functional analyses of murine mitral valves were completed. Results: Loss of actin architecture and collagen compaction capability were seen in CFs deficient of DCHS1 and/or LIX1L. Delivery of a peptide that disrupted the LIX1L-SEPT9 binding domain likewise destroyed actin architecture and reduced ECM compaction. In-vivo epistasis studies support these findings as mitral valve enlargement was exacerbated in DCHS1/LIX1L compound heterozygote mice compared to DCHS1 and LIX1L heterozygotes. Echocardiography of 11-month DCHS1/LIX1L compound heterozygote mice revealed pronounced MVP and increased left atrium diameter relative to DCHS1 and LIX1L heterozygotes. Filamentous SEPTIN9 localized to the actin ultrastructure and was also lost with disturbance of the DLS complex. Nuclei stretch and chromatin compaction were reduced in DCHS1/LIX1L compound heterozygote CFs. Further, histone H4 modifications were observed in DCHS1 KO mouse embryonic fibroblasts that indicate reductions in gene expression. Conclusion: DCHS1, through its interaction with the septin cytoskeleton, must regulate and stabilize actin dynamics in mitral VICs leading to proper valve development. Non-surgical interventions for MVP may be identified through establishing the downstream effects of DCHS1 mutations in our model.